Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett., 1967, 37, 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A., 1978, 75, 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy. One such group of chemical modifications includes bicyclic nucleosides wherein the furanose portion of the nucleoside includes a bridge connecting two atoms on the furanose ring thereby forming a bicyclic ring system. Such bicyclic nucleosides have various names including BNA's and LNA's for bicyclic nucleic acids or locked nucleic acids respectively.
Various bicyclic nucleosides have been prepared and reported in the patent literature as well as in scientific literature, see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8(16), 2219-2222; Wengel et al., PCT International Application number PCT/DK98/00393 (published as WO 99/14226 on Mar. 25, 1999), filed Sep. 14, 1998; Singh et al., J. Org. Chem., 1998, 63, 10035-10039, the text of each is incorporated by reference herein, in their entirety. Examples of issued US patents and published applications include for example: U.S. Pat. Nos. 7,053,207, 6,794,499, 6,770,748 and 6,268,490 and published U.S. applications 20040219565, 20040014959, 20030207841, 20040192918, 20030224377, 20040143114, 20030087230 and 20030082807, the text of each is incorporated by reference herein, in their entirety.
Various carbocyclic bicyclic nucleosides have been prepared and reported in the literature. Such carbocyclic bicyclic nucleosides include 4′-(CH2)3-2′ bridged analogs (see Frier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443); 4′-CH═CH—CH2-2′ bridged analogs (see Albaek et al., J. Org. Chem., 2006, 71, 7731-7740); and 4′-CH2—C(═CH2)-2′ bridged analogs and related analogs (see published International Application WO 2008/154401, published on Dec. 8, 2008).
Bicyclic nucleosides comprising 4′-CH2 (R)-2′, where R is amino or substituted amino, have also been prepared and incorporated into oligonucleotides (see Wengel et al. Chem. Commun., 2003, 2130-2131).
Various other bicyclic nucleosides have been previously reported in the literature including: 4′-(CH2)3—O-2′ and 4′-(CH2)2—O-2′ (and analogs thereof see Morita et al., Biorg. Med. Chem., 2003, 11, 2211-2226; Kaneko et al., published U.S. Patent Application, US 2002/0147332; Japanese Patent Application, HEI-11-33863, published Feb. 12, 1999 and U.S. Pat. No. 7,034,133); 4′-CH2—N(OCH3)-2′ (and analogs thereof see published International Application WO 2008/150729, published on Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′,4′-CH2—N(CH3)—CH2-2′ and 4′-(C═O)—NH—CH2-2′ (and analogs thereof see published U.S. Patent Application, US 2004/0171570; and U.S. Pat. Nos. 6,670,461 and 6,794,499); 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); and 4′-CH2—O—CH2-2′ (and analogs thereof see U.S. Pat. No. 6,403,566, issued on Jun. 11, 2002).
Carbocyclic bicyclic nucleosides have also been prepared and incorporated into oligonucleotides which were further evaluated in biochemical studies including 4′-(CH2)n—C(H)(CH3)-2′ bridged analogs where n is 1 or 2 and analogs thereof (see Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379 and Chattopadhyaya published International Application WO 2008/111908); and 4′-(CH2)3-2′ bridged analogs (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134).
Other bicyclic nucleosides that have been prepared and incorporated into oligonucleotides which were further evaluated in biochemical studies including 4′-(O—CH2)2-2′, 4′-C(H)(CN)-2′ and 2′-(CH2)2—N(R)-4′ bridged analogs (see Imanishi et al., Bioorg. Med. Chem., 2006, 14, 1029-1038 and Chattopadhyaya et al., J. Am. Chem. Soc., 2006, 128, 15173-15187).
Oligonucleotides incorporating 5′-(S)—CH3 bicyclic nucleosides having a 4′-CH2—O-2′ bridging group have been prepared and evaluated in biochemical studies (see commonly owned U.S. Pat. No. 7,547,684, issued on Jun. 16, 2009).
Oligonucleotides incorporating 5′-(S)—CH3 bicyclic nucleosides having a 4′-C(H)(CH3)—O-2′ bridging group have been previously prepared (see commonly owned U.S. Pat. No. 7,666,854, issued on Feb. 23, 2009; and commonly owned International Application PCT/US2009/066863, filed Dec. 4, 2009).